Effects of temperature and light on non-race-specific resistance of potato leaflets to late blight

Effects of temperature and illumination on colonization by Phytophthora infestans of detached leaflets of five potato cultivars differing in field resistance to blight were investigated using an ELISA system to quantify the pathogen. Leaflets of cvs Teena and Shelagh, and in one experiment cv. Brodick, were more resistant to colonization when infected leaflets were incubated at 10°C than at 20°C, but temperature conditions before inoculation had little effect. Both photoperiod and light intensity during illumination of intact plants before inoculation interacted with genotype to determine subsequent colonization of infected leaflets incubated in darkness. Leaflets from plants of cv. Teena were more resistant to colonization after exposure to low, rather than to high, light intensities but photoperiod had no apparent effect. Leaflets of cv. Shelagh grown in a 20 h day were more resistant than those grown in a 10 h day but light intensity had no effect. Leaflets of cv. Brodick were more resistant after a 20 h day than a 10 h day and after exposure to low, rather than to high, light intensities. Leaflets of cv. Bintje were extensively colonized and those of cv. Torridon remained relatively resistant to colonization, irrespective of temperature and lighting conditions. There was a high level of unexplained variation in all the experiments.     

Development ofClonostachys rosea and interactions withBotrytis cinerea in rose leaves and residues

The effect of microclimate variables on development ofClonostachys rosea and biocontrol ofBotrytis cinerea was investigated on rose leaves and crop residues. C.rosea established and sporulated abundantly on inoculated leaflets incubated for 7–35 days at 10°, 20° and 30°C and then placed on paraquat—chloramphenical agar (PCA) for 15 days at 20°C. On leaflets kept at 10°C, the sporulation after incubation on PCA increased from 60% to 93% on samples taken 7 to 21 days after inoculation, but decreased to 45% on material sampled after 35 days. A similar pattern was observed on leaves incubated at either 20° or 30°C. The sporulation ofC. rosea on leaf disks on PCA was not affected when the onset of high humidity occurred 0, 4, 8, 12 or 16 h after inoculation. However, sporulation was reduced to 54–58% on leaflets kept for 20–24 h under dry conditions after inoculation and before being placed on PCA. The fungus sporulated on 68–74% of the surface of leaf disks kept for up to 24 h at high humidity after inoculation, but decreased to 40–51% if the high humidity period before transferral to PCA was prolonged to 36–48 h. The growth ofC. rosea on leaflets was reduced at low inoculum concentrations (10³ and 10⁴ conidia/ml) because of competition with indigenous microorganisms, but at higher concentrations (10⁵ and 10⁶ conidia/ml) the indigenous fungi were inhibited. Regardless of the time of application ofC. rosea in relation toB. cinerea, the pathogen’s sporulation was reduced by more than 99%. The antagonist was able to parasitize hyphae and conidiophores ofB. cinerea in the leaf residues. AsC. rosea exhibited flexibility in association with rose leaves under a wide range of microclimatic conditions, and in reducingB. cinerea sporulation on rose leaves and residues, it can be expected to suppress the pathogen effectively in rose production systems.     

The role of phytoalexins in resistance to fusarium wilt in pigeon pea (Cajanus cajan)

Mechanisms of resistance to fusarium wilt (Fusarium udum) were investigated in pigeon pea cultivars from Malawi. Wilt-susceptible (Malawi local) and wilt resistant (ICP 9145) plants were stem-inoculated with a spore suspension containing 2·10⁶ conidia/ml of the pathogen. Occlusion of a small proportion of infected vessels was observed, but the resistant reaction appeared to depend mainly on rapid phytoalexin synthesis. Four fungitoxic isoflavonoid phytoalexins—hydroxygenistein, genistein, cajanin and cajanol-were isolated from plants 15 days after inoculation. Cajanol was identified as the main antifungal compound. The concentration of cajanol was 329·4 μg/g in the resistant cultivar as against 88·6 μg/g in the susceptible cultivar 15 days after inoculation. Crude extract from the resistant plants sampled at 24 h after inoculation contained 34·8 μg ml of cajanol. The LD₅₀ value of cajanol for spore germination was determined as 35μg/ml. The cajanol content of fungus-infected ICP 9145 10 days after inoculation totally inhibited conidial germination of F. udum.     

Movement of phorate and metabolites from treated leaflets to aphid colonies on broad bean plants

Aphid colonies were established on one second-node leaflet of young broad bean plants and sublethal treatments of ¹⁴C-labelled phorate were applied to the adjacent leaflets. After 3 days, the amounts of toxic and non-toxic ¹⁴C-labelled compounds in the aphids, their honeydew and the untreated foliage were determined. There were no significant differences between the amounts in leaves at the same level on the plant when infested and aphid-free plants were compared, but the aphids and their honey-dew contained two and 23 times as much of the toxic and non-toxic ¹⁴C-compounds, respectively, found in the host leaflets. Following translocation to aphids on leaves above or below treated leaflets, the aphid colonies again contained more labelled compounds than the host-plant leaves. The movement of non-toxic compounds into the roots was reduced when the aphid colonies were situated on foliage between the site of treatment and the roots. More of the toxic and the non-toxic fractions were translocated downwards from the third to the second leaf than in the reverse direction.     

Fusarium stem and fruit rot of sweet peppers in the glasshouse

A stem and fruit rot of sweet peppers ( Capsicum annuum ) occurred in 1992 in West Sussex affecting a large proportion of plants of cv. Tasty and to a lesser extent cv. Mazurka. Fusarium solani was isolated from the stem lesions and also from a short distance above and below them. The disease was largely nodal. Distinctive fruit symptoms also occurred. Symptoms were reproduced by inoculation of wounds on the stem. On agar and in a small in vivo test the pathogen was sensitise to benomyl at 2 μg ml. The disinfectants, commercial sodium hypochlorite, Purogene and Tego 51 effectively killed macrospores following exposure for 15 min.     

Isolation, culture and ultrastructure of a xylem-limited bacterium associated with Sumatra disease of cloves

A xylem-limited bacterium (XLB) previously shown to be associated with Sumatra disease of clove trees was consistently isolated from diseased but not from healthy trees. Isolation of the XLB was most successful using freshly expressed bacterial ooze obtained from root and branch tissues with low to moderate levels of xylem vessel invasion in trees at early stages of disease. XLB were commonly isolated from trees with the rapid form of disease but infrequently from those with slow decline. Bacterial colonies grew slowly on nutrient agar, reaching a maximum diameter of 1 mm in 5-7 days at 28^oC. Cells of the bacterium were 0.5-0.6 μm in width and 1.0-1.5 μm in length and did not possess flagella. The bacterial envelope was strongly rippled and Gram negative.     

Effects of humidity and air speed on sporulation of Phytophthora infestans on potato leaves

An apparatus is described for the control of humidity, air speed and temperature around detached potato leaflets and whole plants. Leaflets and plants inoculated with Phytophthora infestans were incubated in air at 15°C. Abundant sporangia were formed in an air speed of 0.3 × 10⁻³ m/s when the ambient humidity was 90–100% RH, but not at 85 or 80% RH. At air speeds of 5.5 × 10⁻³ and 13.7 × 10⁻³ m/s there were numerous sporangia at 100% RH, but not at 95–80% RH. The number of sporangia formed on leaflets kept in air flowing at 5.5 × 10⁻³ m/s with alternating humidities of 80 and 100% RH each for 12 h per day was intermediate between numbers formed on leaflets incubated at each humidity continuously, and higher than numbers formed at a constant 90% RH. Removing leaflets from plants before inoculation did not affect the number of sporangia produced.     

Leaflet abscission and phytoalexin production during the response of two faba bean breeding lines to Botrytis infection

Comparisons were made between two breeding lines of faba bean (designated 224 and 335), following inoculation of leaf material with Botrytis cinerea and the more aggressive B. fabae . Line 335 exhibited greater initial resistance to infection, in terms of retarded lesion development and higher rates of phytoalexin production. However, in whole plant inoculation experiments line 335 abscinded its leaflets more readily after infection. The role of the gaseous plant hormone ethylene in this reaction was analysed. Both lines exhibited a similar abscission response to exogenous ethylene, applied at a concentration of 10 μL L⁻¹. However, there were significant differences in the ability of the lines to produce ethylene. In response to inoculation with B . fabae , line 335 liberated ethylene at a higher concentration and for a longer period. These results suggest that the differential levels of abscission are a consequence of differences in the biosynthesis of ethylene in the two lines rather than in their responses to ethylene.     

Light and Scanning Electron Microscopy Studies on the Infection of Oriental Lily Leaves By Botrytis Elliptica

Light, scanning electron and fluorescent microscopy were used to observe the infection process of Botrytis elliptica on leaves of oriental lily (cv. Star Gazer). At 20 °C and 100% relative humidity, conidia germinated on both adaxial and abaxial foliar surfaces, but germ tubes failed to invade epidermal cells on the adaxial surface. On abaxial surfaces, short (< 20 m) swollen germ tube appressoria penetrated through stomatal openings (19%), through the epidermis near guard cells (52%), or directly through epidermal cells (29%). Esterase activity was detected on germ tubes and conidia after 6 h of incubation, and deformation of the cuticle on abaxial surfaces of lily was observed surrounding infection sites. By 3 h after inoculation, almost 70% of the conidia had germinated, but no penetration was observed. At 6 h after inoculation, almost one-third of germinated conidia had penetrated epidermal cells, and water-soaked lesions were associated with 20% of the penetrations. By 9 h after inoculation, approximately 60% of the germinated conidia had penetrated plant tissues, and water-soaked lesions were associated with 60% of the infections. Fluorescent microscopy with a specific fungal stain allowed assessment of successful infection and visualization of sub-epidermal hyphae. We conclude that penetration of abaxial foliar surfaces of oriental lilies by B. elliptica occurs via short swollen germ tube appressoria mostly near stomata.     

Variation in the early development of Bremia lactucae on lettuce cultivars with different levels of field resistance

The early stages of development of Bremia lactucae (lettuce downy mildew) were examined on lettuce cultivars possessing high (Iceberg and Regina di Maggio) and low (Great Lakes and Plenos) levels of field resistance. Germ tubes, appressoria, penetration, primary and secondary vesicles, intercellular hyphae and haustoria were observed 3. 6 and 24 h after inoculation of cotyledons and of leaf discs from adult plants. Differences were observed between cv. Iceberg and susceptible genotypes in the percentage of spores germinating and the incidence and speed of development of infection structures. Secondary vesicles were first observed 24 h and 6 h after inoculation in Iceberg and susceptible genotypes, respectively. The lowest incidence of secondary vesicle formation 24 h after inoculation (48 and/or 43%) was recorded in Iceberg and Regina di Maggio, and the highest incidence (68%) occurred in Plenos. The formation of intercellular hyphae and haustoria was not observed in cv. Iceberg some 24 h after inoculation. There were significant differences in the lengths of germ tubes formed on different cultivars. Those on cv. Iceberg were longer than those formed on susceptible cultivars. The results indicate that the field resistance of B. lactucae may result from mechanisms which are effective in the early stages of infection.     

The effect of hot-water treatment on the levels of antifungal diene and quiescence of Colletotrichum gloeosporioides in avocado fruits

Quantitative changes in the antifungal compound 1-acetoxy-2-hydroxy-4-Oxo-heneicosa-12, 15-diene in harvested avocado fruits, and the development of symptoms caused by Colletotrichum gloeosporioides , were investigated following treatment with hot water at 55°C for 10 min. The concentration of the compound in the peel and flesh was 2000 and 2600 μg/g fresh weight, respectively, at the time of harvest, but decreased rapidly during the first 24 h. Levels of the diene had substantially recovered after 50 h. However antifungal diene levels in the peel of hot-water-treated fruit did not recover until 98 h. The levels in the flesh were unaffected by the treatment. Following inoculation, hot-water-treated fruits developed clear symptoms after 2 days, whereas untreated fruits showed only minor symptoms after 6 days. If inoculation was delayed by 24, 48 or 72 h after treatment, then symptoms on treated fruits did not develop until the sixth day as observed for untreated fruits. The correlation between the two systems suggests that quiescence is probably maintained by the level of antifungal diene present in the peel at the time of fungal penetration and the formation of a subcuticular hypha.     

Control of potato late blight using controlled-release granules containing ofurace

In an attempt to reduce or eliminate the need for spraying to control potato late blight caused by Phytophthora infestans, investigations were made on the use of controlled-release granules containing the systemic fungicide ofurace. The granules when mixed with sand and buried in soil released the fungitoxicant for about 100 days. When the granules were applied in furrows at planting, protection of potato plants (cv. Maris Bard), assessed by inoculation of detached leaflets with P. infestans, lasted for between 85 and 100 days. Application of the granules when the plants were earthed up 48 days after planting did not result in better late-season protection, possibly due to poorer uptake of the fungitoxicant by the plants at this time. Chemical analysis of leaves from plants that had received in-furrow treatment indicated that ofurace at 0.2–0.5 μg g^?1 fresh weight was needed to confer protection from late blight.     

The virus-vector relationship of the tomato leafcurl virus (TLCV) and its vector,Bemisia tabaci gennadius (Hemiptera: aleyrodidae)

Basic studies carried out in India showed that the incubation period of TLCV in plants varied from 8 days in August to 90 days in winter. The acquisition threshold for the whitefly,Bemisia tabaci Gen., was 31 min; it resulted in 3% transmission. An acquisition access of 24 h for a female whitefly on a TLCV source resulted in 30% transmission. A minimum feeding period of 32 min was required by a viruliferous whitefly to cause infection on tomato test plants; this gave 4% transmission. With inoculation access of 24 h on tomato test plants, the transmission rose to 24%. Starving the vector for 1 h pre-acquisition or 1 h pre-inoculation resulted in higher levels of transmission of TLCV: 36 and 40%, respectively, compared with 20% for non-starved whiteflies. Extending the fasting period beyond 1 h resulted in a reduced transmission level. The whiteflies could acquire the virus from the cotyledonary leaves of an infected tomato plant, with a resultant 28% transmission; but infection did not occur when the whiteflies had an inoculation access to such leaves. Higher transmission rates were obtained when the younger leaves on tomato plants were used for acquisition and inoculation. Transmission was 8 and 38% when five whiteflies per plant were allowed 24 h of acquisition access to 11- and 2-month-old virus sources, respectively. After an acquisition access of 24 h to a TLCV source, male and female whiteflies retained their infectivity for 5 and 53 days, respectively. Nymphs can acquire and transmit the virus. When ten whiteflies of each sex were given 24 h of acquisition and of inoculation access, the subsequent transmission rate of males and females was 56 and 86%, respectively. This virus is not transovarially transmitted. Whitefly colonies raised on brinjal were more efficient (70 and 84% transmission in two experiments) than those raised on chilli, cotton, cowpea, tobacco or tomato.     

Induction of resistance in cocoa against Crinipellis perniciosa and Verticillium dahliae by acibenzolar- S-methyl (ASM)

The benzothiadiazole compound acibenzolar- S -methyl (ASM) was assessed as an inducer of resistance against Crinipellis perniciosa , agent of witches' broom, and Verticillium dahliae , agent of vascular wilt, both on cocoa. ASM induced a reduction in incidence of witches' broom of up to 84·5% when sprayed 30 days before inoculation on cocoa seedlings of cv. Catongo. ASM also induced a reduction in severity of Verticillium wilt to 55·4% on cv. Theobahia. For both pathosystems, effects of dose on disease were not clearly observed. The efficacy of the inducer increased with the interval between sprayings and the respective inoculations with the pathogens. In another experiment, the effect of ASM on the control of witches' broom on cocoa seedlings was compared with that of cuprous oxide and tebuconazole, all sprayed 15 days before inoculation. ASM reduced disease incidence by 60·1% compared with the inoculated control. ASM was superior to tebuconazole, and there was also a tendency for ASM to be better than cuprous oxide. To understand the mechanism of action of ASM as an inducer of resistance, alterations in the levels of total phenolics, polyphenol oxidases and peroxidases were evaluated 3, 15 and 30 days after spraying of seedlings of cv. Catongo. Enzyme activities from seedlings of cv. Theobahia were evaluated 30 days after spraying. On cv. Catongo, no significant differences in total phenolic content and polyphenol oxidase activity were detected after spraying. However, an increase in peroxidase activity was detected at all times of evaluation. On cv. Theobahia, significant increases in activities of peroxidase and polyphenol oxidase were detected, indicating that defence responses due to ASM were dependent on host genotype.     

Biochemical responses of coffee resistance against Meloidogyne exigua mediated by silicon

Coffea arabica cultivars Catuaí 44 and IAPAR 59, susceptible and resistant, respectively, to the root knot nematode Meloidogyne exigua, were grown in pots containing Si‐deficient soil amended with either calcium silicate (+Si) or calcium carbonate (?Si). There was an increase of 152 and 100%, respectively, in Si content of root tissue of cvs Catuaí 44 and IAPAR 59 in the +Si compared to the ?Si treatment, but no significant difference between Si treatments for calcium content. Plants, assessed 150 days after inoculation (d.a.i.) showed that the number of galls (NG) and number of eggs (NE) significantly decreased by 16·8 and 28·1% respectively, for susceptible cv. Catuaí 44 in the presence of Si, whilst both NG and NE were significantly lower for cv. IAPAR 59 compared to the susceptible cultivar regardless of Si treatments. In a separate experiment, biochemical assays were carried out 5 and 10 d.a.i. There was no significant difference between Si treatments and cultivars for concentration of total soluble phenolics. The concentration of lignin‐thioglycolic acid (LTGA) derivatives significantly increased by 11·5% in roots of nematode‐inoculated plants of susceptible cv. Catuaí supplied with Si. In roots of inoculated plants of resistant cv. IAPAR 59, the increase was 23 and 10%, respectively, for treatments with and without silicon. Peroxidase (POX), polyphenoloxidase (PPO) and phenylalanine ammonia lyase (PAL) activities significantly increased in roots of inoculated plants compared with roots of non‐inoculated plants, regardless of cultivar or Si treatment. In +Si treatments at 10 d.a.i., POX activity in roots of nematode‐inoculated plants of cvs Catuaí 44 and IAPAR 59 increased by 39·9 and 31·3%, respectively; PPO increased by 54·9 and 56·1%; and PAL activity was also higher at 26·6 and 62·9%. It was concluded that supplying Si to coffee plants increases root resistance against M. exigua by decreasing its reproductive capacity.     

Host status effect of cowpea and sunflower on the populations ofMeloidogyne javanica andRotylenchulus reniformis

A highly susceptible cowpea,Vigna sinensis cv. Baladi plants were tested as trap plants for eitherMeloidogyne javanica orRotylenchulus reniformis under greenhouse conditions. The plants were gathered by cutting them above the surface of the soil or by uprooting them, 1/2, 1, 3, 6, 12, 24, 36 and 48 days after nematode inoculation. Both of the mentioned nematodes began to mature and lay eggs after the 12th day from their inoculation. Hence, it is advised to pull up cowpea plants from 3–12 days after nematode inoculation. After planting sunflower,Helianthus annus cv. Miak replacing cowpea, the nematode populations were higher, in most cases, on sunflower plants replacing cutting cowpea than those on sunflower replacing uprooted cowpea. The highest percentages of nematode reduction were 98.55 and 99.57 forM. javanica and 95.09 and 92.90% forR. reniformis on sunflower plants replacing cutting and uprooted cowpea plants after 12 days from nematode inoculation, respectively.M. javanica andR. reniformis decreased the length and weight of sunflower plants as affected by planting time and method of cowpea harvest. This method of nematode control is cheaper, easy and pollution free.     

Effect of metalaxyl on formation and germination of oospores of Phytophthora infestans

Oospores of Phytophthora infestans were produced in potato leaf discs floating on metalaxyl solution (100 μg mL⁻¹ a.i.) and inoculated with all combinations of two metalaxyl-sensitive and two -resistant parental isolates. Numbers of oospores produced varied between different matings, depending on parents, in the absence of the fungicide and when metalaxyl was added 0, 7, 14 and 21 days after inoculation. Oospores were not produced when metalaxyl was added at the time of inoculation (0 days) when either one or both parents were sensitive to metalaxyl. In two of three such matings further oospore formation was arrested when metalaxyl was added either 7 or 14 days after inoculation. Oospores extracted from leaf discs 14, 21 and 28 days after inoculation were assessed for germination on water agar after 21 days. Germination of oospores from water control treatments varied between 6 and 30% depending on the cross. Germination was significantly reduced in oospores of metalaxyl-sensitive parents extracted 28 days after inoculation of leaf discs treated with metalaxyl 0, 7 and 14 days after inoculation compared with the 21-day treatment. Minimal differences in germination were observed for oospores from the mating of resistant parents irrespective of metalaxyl treatment, although germination was generally low, not exceeding 8.5%.     

Microscopical studies of resistance to powdery mildew disease in the hop cultivar Wye Target

Microscopical studies of fungal development and host responses during infection of the hop cultivars Northern Brewer (susceptible) and Wye Target (resistant) with a Zenith isolate of Sphaerotheca humuli are described. Resistance to powdery mildew disease in cv. Wye Target is principally determined by the R₂ major gene. Fungal growth was typically restricted in cv. Wye Target following formation of a single haustorial initial or haustorium which failed to develop the characteristic lobes found in susceptible cells. The hypersensitive reaction of penetrated epidermal cells was associated with death of haustorial initials but the associated appressorium remained alive during the first 2 days after inoculation. In leaves expressing resistance, histochemical staining revealed deposition of lignin-like material and callose in penetrated cells and to a lesser extent in underlying palisade mesophyll cells. Transmission electron microscopy and enzymic digestion clearly demonstrated extensive paramural deposition of β-1.3 glucans (callose) in reacting mesophyll cells. Plant cell death, lignification and widespread callose deposition were rarely observed at infection sites in cv. Northern Brewer but collars of callose were deposited around the necks of all haustoria formed.     

Prior Inoculation with Non-pathogenic Fungi Induces Systemic Resistance to Powdery Mildew On Cucumber Plants

A spray inoculation of the first leaf of 2-leaf stage cucumber plants with a non-pathogenic isolate of Alternaria cucumarina or Cladosporium fulvum before a challenge inoculation with the pathogen Sphaerotheca fuliginea induced systemic resistance to powdery mildew on leaves 2–5. Systemic resistance was expressed by a significant (p < 0.05) reduction in the number of powdery mildew colonies produced on each leaf of the induced plants, as compared with water-sprayed plants. Systemic resistance was evident when a prior inoculation with each of the inducing fungi was administered 1, 3 or 6 days before the challenge inoculation with S. fuliginea. Increasing the inoculum concentration of A. cucumarina or C. fulvum enhanced the systemic protection and provided up to 71.6% or 80.0% reduction, respectively, in the number of colonies produced on upper leaves, relative to controls. Increasing the inoculum concentration of S. fuliginea used for challenge inoculation, increased the number of powdery mildew colonies produced on both induced and non-induced plants. Pre-treated plants, however, were still better protected than controls, indicating that the level of systemic protection was related to the S. fuliginea inoculum concentration. The induction of systemic resistance against powdery mildew by biotic agents, facilitates the development of a wide range of disease management tools.     

Behavior and mutation of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in <Emphasis Type="Italic">Solanum toxicarium</Emphasis> grown in aseptic culture

To study the behavior and mutation of Ralstonia solanacearum in Solanum toxicarium, which is resistant to bacterial wilt, S. toxicarium was grown in aseptic culture and inoculated with R. solanacearum. Although 60%–80% of the inoculated plants were wilting after 2 to 3 days, most wilted plants had recovered by 20 days after inoculation. The pathogen was reisolated from over 98% of inoculated plant stems, but the percentage of recovery decreased the closer the isolation sites were toward the upper stem sections. Three colony types, characterized as fluidal white, nonfluidal red, and a mixture of fluidal white and nonfluidal red, were reisolated from the stems. Nonfluidal red colonies were less virulent on tomato plants than fluidal white colonies.